Oxidative stress is one of the major contributing factors in ethanol (alcohol)-mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells is being produced through direct inhibition of the mitochondrial respiratory chain and induction or activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase, and NADPH-oxidase. Despite the well-established roles of ROS/RNS in alcohol-induced cellular dysfunction and injury, it is poorly understood which proteins are oxidatively-modified by elevated ROS/RNS and whether their functions are altered. To address these questions, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to positively identify oxidized proteins in alcohol-exposed hepatoma cells or animal tissues. During this fiscal year, we have applied this targeted proteomics approach to identify oxidized proteins in cytosolic and mitochondrial fractions of alcohol-exposed mouse or rat livers. The biotin-NM labeled oxidized proteins were purified with streptavidin-agarose beads and resolved by 2-D gel electrophoresis. Protein spots, that displayed differential abundances in alcohol-fed mouse or rat livers compared to those in the pair-fed controls, were excised from the 2-D gels, in-gel digested with trypsin and subjected to mass spectrometry. Mass spectrometric data revealed that many cytosolic proteins involved in chaperone activities, anti-oxidant defense, intermediary metabolism including the transmethylation pathway and cytoskeletal proteins were oxidized in alcohol-fed mouse livers. Our current results are likely to explain the underlying mechanisms for the inactivation of some of these enzymes leading to the reduced levels of antioxidants such as S-adenosylmethionine and glutathione with the increased levels of homocysteine observed in alcohol-exposed animal tissues and alcoholic human subjects. Oxidative inactivation of anti-oxidant enzymes such as catalase and peroxiredoxin through sulfinic/sulfonic acid formation of its active site Cys may contribute to the elevated levels of peroxides observed in CYP2E1-containing E47 HepG2 hepatoma cells and alcohol-exposed animals. Many mitochondrial proteins involved in mitochondrial electron transfer, energy production, beta-oxidation of fatty acids, and chaperone activities were also oxidatively-modified in alcohol-exposed rat livers. For instance, mitochondrial 3-ketoacylCoA thiolase involved in the beta-oxidation of fatty acids was oxidatively-modified and inactivated in alcohol-fed rats, compared to that in the pair-fed control rats. Inhibition of 3-ketoacylCoA thiolase and three other enzymes in the mitochondrial beta-oxidation pathway is consistent with increased accumulation of triglycerides determined by biochemical and histological methods. Our immunoblot analysis with the antibody against 3-nitroTyr also showed that tyrosine (Tyr) residues of mitochondrial ATP synthase (complex V) were nitrated in alcohol-exposed animals, resulting in its inhibition and subsequently reduced ATP production. Nitration of the active site Tyr residues of ATP synthase was further confirmed by mass spectral analysis. Furthermore, mitochondrial aldehyde dehydrogenase (ALDH2) involved in the metabolism of acetaldehyde and toxic lipid peroxides were oxidized, leading to inhibition of its activity. To directly demonstrate oxidative modification of ALDH2 in alcohol-fed rats, we immunopurified the mitochondrial ALDH2 protein from pair-fed control rat livers or alcohol-fed rats. One immunopurified ALDH2 protein was identified for both samples. However, S-nitrosylated protein, detected with the anti-S-nitrosylated-Cys antibody, was observed only in the alcohol-treated rats but not in control animals. Addition of a reducing agent such as DTT caused disappearance of the S-nitrosylated-Cys band in alcohol-fed rats. These data strongly suggest that the active site Cys302 of ALDH2 was reversibly S-nitrosylated in alcohol-fed rats. Similar results about oxidative modification of ALDH2 were also observed in ethanol-exposed hepatoma cell lines. These results on S-nitrosylation of ALDH2 may also explain the underlying mechanism for the reduced levels of ALDH2 activity often observed in alcoholic individuals and after exposure to various toxic chemicals. Collectively, based on the current results, inactivation of many of these oxidized mitochondrial proteins is likely to contribute to alcohol-induced mitochondrial dysfunction and increased sensitivity toward ethanol-mediated oxidative tissue injury. [unreadable] [unreadable] In addition, we have investigated the signaling mechanism during cellular damage caused by many toxic compounds. Our earlier results showed selective and persistent activation of c-Jun N-terminal protein kinase (JNK) by many toxic substrates of CYP2E1 such as acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids. In contrast, ethanol, another substrate of CYP2E1, and non-CYP2E1 substrates such as troglitazone, hydrogen peroxide, etoposide, and staurosporine (STS) activated JNK and p38 protein kinase (p38 kinase) simultaneously. Our results also showed that all these compounds caused translocation of proapoptotic Bax from cytoplasm to mitochondria in a time-dependent manner. Since the mechanism of Bax activation and its mitochondrial translocation prior to apoptosis was unknown, we further investigated the mechanism for Bax activation after cells were treated with various cell death stimulants. Our results showed that stress-activated protein kinases stimulated after exposure to various toxic compounds directly phosphorylated Bax before it was translocated to mitochondria to initiate actual cell death observed at later time points. Phosphorylation of Bax was demonstrated by the shift of the pI value of 4.0 (phosphorylated Bax) from pI 5.1 (non-phosphorylated Bax) on 2-D gels and confirmed by metabolic labeling with 32P-inorganic phosphate. Important roles of JNK and/or p38 kinase in mitochondrial translocation of Bax and apoptosis were demonstrated by using a specific inhibitor of JNK or p38 kinase and a specific siRNA to MAPKK4, the upstream kinases of JNK and/or p38 kinase. Pretreatment with each agent significantly reduced the activity of each kinase and the rates of mitochondrial translocation of Bax and apoptosis. To determine the phosphorylated amino acid, several Bax mutants (Ser87Ala, Thr167Ala and others) were prepared, based on the fact that JNK and p38 kinase are proline-directed protein kinases. Critical roles of phosphorylation of Bax in its mitochondrial translocation were further confirmed by confocal microscopy of various Bax mutants and transfection into Bax/Bak double knockout mouse embryonic fibroblast cells. Our confocal microscopic results with various Bax mutants clearly show that Thr167 is a critical amino acid which is phosphorylated by stress-activated protein kinases. Taken together, our results suggest that JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation, which might disrupt the previous interaction between the N-terminal domain and the C-terminal transmembrane domain of Bax. Exposure of the C-terminal transmembrane domain is likely to lead to mitochondrial translocation of Bax to initiate mitochondria-dependent apoptosis. Our results demonstrate for the first time that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis. Taken together, our results are likely to explain the underlying mechanisms for the positive relationship between activation of JNK or p38 kinase and apoptosis caused by many distinct cell death stimulants or conditions, as previously reported by many scientists.